Spectroscopy is a method for obtaining information on a molecular scale by the use of light. This information can be related to the rotational, vibrational and/or electronic states of the molecules probed as well as dissociation energy and more. The rotational and/or vibrational spectrum of a given molecule is specific for that molecule. As a consequence, molecular spectra in particular rotation and/or vibrational spectra are often referred to as ‘fingerprints’ related to a specific molecule. Information related to rotational, vibrational and/or electronic states of molecules can therefore be used to analyze a sample comprising a number of unknown molecular components, thereby obtaining knowledge about the molecular components in the sample.
The basis for a spectroscopic setup is a light source, e.g. a laser, which is used for illuminating a sample. The light from the light source (the incoming light) will interact with the sample, and often result in an alternation of the light which is transmitted through, emitted by, reflected by and/or scattered by the sample. By collecting the altered light and analyzing its spectral distribution, information about the interaction between the incoming light and the molecular sample can be obtained; hence information about the molecular components can be obtained.
The spectral distribution is typically measured by using a spectrometer. A spectrometer is an optical apparatus that works by separating the light beam directed into the optical apparatus into different frequency components and subsequently measuring the intensity of these components by using e.g. a CCD detector, a CCD array, photodiode or such.
The altered light reflecting interactions between the incoming light and the molecular sample can roughly be characterized as either emission or scattering. The emission signals have relatively broad spectral profiles as compared to scattering light signals, which normally display quite narrow spectral lines. One process often dominates over the other, but both processes can and most often will occur simultaneously. The intensity of the emitted light vs. the intensity of the scattered light depends among other things on the frequency and the power of the incoming light, the intensity of the incoming light at the measuring point in the sample, and the molecular components in the sample.
Scattered light can be classified as being either elastic or inelastic and these are characterized by being spectroscopically very narrow signals. Elastic scattering is referred to as Rayleigh scattering, in which there is no frequency shift. Rayleigh scattering thus has the same frequency as that of the incoming light.
The most commonly known example of inelastic scattering is Raman scattering, in which there is an energy interchanging between the molecule and the photons of the incoming light. The frequencies, i.e. the spectral distribution of the Raman scattered light will be different from that of the incoming light and uniquely reflect the specific vibrational levels of the molecule; hence it is a fingerprint spectrum. This can be used for identification of the molecular composition of the substance probed and/or the concentration of the specific molecules in the substance.
Raman scattering is a relatively weak process compared to e.g. Rayleigh scattering and fluorescence. Reduction of contributions from these other processes is thus desirable when collecting Raman scattered light. In addition, the intensity of the Raman scattered light depends strongly on the frequency and the intensity of the incoming light. If these are variable, it may therefore be essential to monitor power fluctuations in the incoming light if one is to receive reliable information about the distribution of molecular components in different samples and/or sample spot bases on analysis of the collected Raman scattered light, depending on the precision needed. The same is true if the analysis of the molecular components in a sample and/or different sample spots is bases on emission spectra.
Skin comprises a number of layers having different characteristics and containing different kinds of cells and structures. Various proposals for using Raman spectroscopy to measure glucose in skin or in other parts of the body have been made, but none of these has to date provided a system which can be used on most candidate subjects without adjustment to suit a particular individual and without calibration for that individual. It is thereby possible to calibrate an instrument against measurements of blood glucose concentration made on one individual or a group of individuals by other means such as chemical analysis and to apply that same calibration when the instrument is used on other individuals than the one or ones involved in the calibration. We have now appreciated that the key to achieving such a result is to ensure that the Raman scattered light that is collected for measurement originates at or close to a specific depth within the skin.
Caspers et al; Biophysical Journal, Vol 85, July 2003, describes an in vivo confocal Raman spectroscopy method and apparatus which is said to be useful for measuring glucose. It contains however no instruction as to the depth from which the Raman scattering should be collected in a glucose measurement and there is a strong suggestion deducible from the teaching that the apparatus had not actually been tried for this purpose.
WO2008/052221 describes a method and apparatus for coherent Raman spectroscopy that transmits light through a sample surface such as skin and tissue to a focal plane within the sample to measure for instance glucose. However, no teaching is present of the importance of selecting a particular depth for the focal plane or where this should be. Indeed, it is specifically acknowledged that using the described apparatus variations in the detected signal occur when the analyte concentration is constant due to effects of skin temperature and hydration. No suggestion is present that such effects can be avoided by a careful selection of the depth from which the measurements are taken.
WO97/36540 describes determination of the concentration of e.g. glucose using Raman spectroscopy and an artificial neural network discriminator. However, the Raman signals are not selectively obtained from a particular depth and the need to compensate for non-linearities arising from signals penetrating to a depth of >500 μm is discussed.
WO00/02479 discloses a method and apparatus for non-invasive glucose measurement by confocal Raman spectroscopy of the aqueous humor of the anterior chamber of the eye. Naturally, there is no teaching of a depth at which to make optimal measurements in skin.
WO2009/149266 refers back to Ermakov I V, Ermakova M R, McClane R W, Gellermann W. Opt Lett. 2001 Aug. 1; 26(15):1179-81, ‘Resonance Raman detection of carotenoid antioxidants in living human tissues.’ which describes using resonance Raman scattering as a novel noninvasive optical technology to measure carotenoid antioxidants in living human tissues of healthy volunteers. By use of blue-green laser excitation, clearly distinguishable carotenoid Raman spectra superimposed on a fluorescence background are said to be obtained.
Chaiken et al (Noninvasive blood analysis by tissue modulated NIR Raman spectroscopy, J. Chaiken et. al., Proc. of SPIE optical Eng., 2001, vol. 4368, p. 134-145) obtained a correlation of only 0.63 between Raman based measurements and fingerstick blood glucose measurements across several individuals, but were able to obtain a correlation of 0.90 for a single individual. The setup utilized by Chaiken et al comprises a collimated exitation beam and so naturally do not disclose any optimal focal depth.
WO2006/127766, WO02/07585 and US2006/0234386 all describe the use of Raman spectroscopy for measuring lactate through the skin surface. Lactate measurements may be used for various purposes including monitoring the effect of exercise and determining whether a person has died or is still living. In critical care, the monitoring of blood lactate is of importance. High levels of lactate may be associated with myocardial infarction, cardiac arrest, circulatory failure and emergency trauma situations.